Glucose-6-phosphate dehydrogenase (G6PD), the first enzyme in the pentose phosphate pathway, serves as an antioxidant enzyme by supplying NADPH to maintain intracellular glutathione stores. The importance of G6PD as an antioxidant is demonstrated by G6PD- deficient erythrocytes that hemolyze when subjected to oxidant stress. The role of G6PD as an antioxidant enzyme in cells with alternative sources of NADPH has recently been established by inhibiting G6PD and demonstrating a enhanced susceptibility to reactive oxygen species (ROS). The role of G6PD as an antioxidant enzyme in cells with alternative sources of NADPH has recently been established by inhibiting G6PD and demonstrating an enhanced susceptibility to reactive oxygen species (ROS). The role of G6PD in the oxidant defense of vascular cells, which are often exposed to high levels of ROS in vascular disease, has not yet been studied. Preliminary data presented in this application suggest that G6PD modulates oxidant stress in vascular smooth muscle cells (VSMC) and that G6PD deficiency may adversely effect these homeostatic mechanisms and influence vascular responses. In the first two years of this proposal, the applicant will study the role of G6PD in antioxidant biochemistry and the molecular techniques required to alter G6PD expression with the assistance of Dr. Robert Stanton and Dr. Yingyi Zhang who have expertise in these fields. During this time, the applicant will specifically address the first aim of this proposal; namely, to define the role of G6PD as an antioxidant enzyme in VSMC subjected to oxidant stress. In the latter three years the applicant will begin a period of independent investigation further examining the role of G6PD in VSMC functional responses to oxidant stress with the sponsor of this proposal, Joseph Loscalzo, M.D., Ph.D. During this period, the applicant: 1) characterize the role of G6PD on viability, proliferation and apoptosis in VSMC subjected to ROS, and 2) examine the role of G6PD on viability, proliferation and apoptosis in VSMC subjected to ROS, and 2) examine the role of G6PD in maintaining bioavailable nitric oxide (NO) levels and NO-mediated responses in VSMC exposed to ROS. The work contained in this proposal will provide new insights into the role of G6PD as an antioxidant enzyme in vascular cells. Through completion of this project, the applicant expects to acquire the fundamental research skills to become established as an independent investigator focused on cellular redox state, antioxidant function, and vascular homeostasis.